Self-incompatibility, a mechanism that prevents self-fertilization in flowering plants, is based on the ability of the pistil to recognize the presence of self-pollen and on the female tissue's capacity to restrict the growth or germination of self-related but not of genetically unrelated pollen. Although the mechanism of self-recognition is not yet known, a chromosomal location, the S-locus, has been shown from genetic crosses to encode the putative pistil and pollen elements that interact in this recognition process. When both the captured pollen grain and the receptive pistil possess and express different S-locus haplotypes, pollen growth proceeds; when the two S haplotypes are identical, pollen growth, as well as self-pollination, are prevented. The expression of S-locus encoded elements in two tissues, the female sporophytic tissue as well as in the anther or pollen (or in both male tissues), is a hallmark of all models of self-incompatibility and distinguishes gene regulation in this system from many other systems in which tissue specificity is limited to a single location.
Self-incompatibility has been best studied in the Brassicaceae and Solanaceae, and genes associated with the S-locus have been identified in species of Brassica, Nicotiana, Petunia, and Solanum. In Brassica, two partially homologous genes have been shown to reside at the S-locus: one, SLG, encodes a secreted glycoprotein that is present in stigma papillar cells and in the tapetum and microspores of the anther, and the other, SRK, specifies a putative membrane-spanning receptor protein kinase that is present in Brassica pistil and anther. Expression of the S-locus products in the Brassicas is controlled sporophytically. A haploid pollen grain will possess a self- incompatibility phenotype that is determined by the two S-locus alleles carried by the parent plant. In contrast, the self-incompatibility phenotype of pollen derived from plants that express gametophytic control of self-incompatibility (e.g., in the Solanaceae) is dictated by the single S-locus haplotype carried by the pollen.
Evidence for this specificity of SLG promoter activity derives from genetic ablation studies in which a chimeric gene construct consisting of the SLG promoter fused to the diphtheria toxin subunit A (DTA) gene was introduced into tobacco (M. K. Thorsness, M. K. Kandasamy, M. E. Nasrallah and J. B. Nasrallah, Dev. Biol., Vol. 143, (1991) pages 173-184), Brassica (M. K. Kandasamy, M. K. Thorsness, S. J. Rundle, J. B. Nasrallah and M. E. Nasrallah, Plant Cell, Vol. 5, (1993) (in press)), and Arabidopsis (M. K. Thorsness, M. K. Kandasamy, M. E. Nasrallah and J. B. Nasrallah, Plant Cell, Vol. 5, (1993) (in press)). Transformation of these plants with the SLG-DTA gene fusion resulted in the production at high frequency of transgenic plants that underwent normal differentiation and produced flowers in which only specific cells of the pistil and anther were ablated.
Surprisingly, when the Brassica SLG gene is introduced into Nicotiana tabacum, a self-compatible species belonging to the Solanaceae family, it is expressed in pistils in a manner similar to that noted for the S-linked RNase of Nicotiana alata and not according to the pattern shown for this gene in Brassica. In transgenic tobacco, the product of the introduced Brassica SLG gene accumulates in the secretory zone of the stigma, the transmitting tissue of the style, and to a lesser degree in the placental epidermis of the ovary. In Brassica, endogenous SLG molecules are detected mainly in the cell wall of stigma papillar cells. These expression patterns are consistent with the site and timing of self-pollen rejection in Brassica and Nicotiana.
Similar findings have been made with regard to S-locus expression in the anther. The analysis of the SLG-DTA fusion and of a reporter gene fusion consisting of the SLG promoter fused to the reporter .beta.-glucuronidase (GUS) gene have identified the cell types of the pistil and anther in which the SLG promoter is active. In the pistils of transgenic Brassica and Nicotiana, the promoter is active in cells of the stigma and in the transmitting tissue of the style and ovary (T. Sato, M. K. Thorsness, M. K. Kandasamy, T. Nishio, M. Hirai, J. B. Nasrallah and M. E. Nasrallah, Plant Cell, Vol. 3, (1991) pages 867-876; M. K. Thorsness, M. K. Kandasamy, I. E. Nasrallah and J. B. Nasrallah, Dev. Biol., Vol. 143, (1991) pages 173-184). In the anthers of transgenic Brassica, promoter activity is evident in the tapetum, a sporophytic tissue of the anther, and in microspores (T. Sato, M. K. Thorsness, M. K. Kandasamy, T. Nishio, M. Hirai, J. B. Nasrallah and M. E. Nasrallah, Plant Cell, Vol. 3, (1991) pages 867-876). In transgenic tobacco anthers on the other hand, the SLG-GUS fusion exhibits strict gametophytic expression: GUS activity is detected in pollen grains and not in the sporophytic tissues of the anther (M. K. Thorsness, M. K. Kandasamy, M. E. Nasrallah and J. B. Nasrallah, Dev. Biol., Vol. 143, (1991) pages 173-184). Moreover, approximately one half of the pollen grains of transformed plants that contain a single copy of the introduced gene show GUS activity. On the other hand, Brassica plants transformed with this construction display GUS activity in the tapetum, a sporophytic tissue of the anther, and in pollen microspores. Thus, although the S-locus-linked genes identified to date differ in plants possessing sporophytic and gametophytic forms of self-incompatibility, a common, conserved mechanism apparently exists in Brassica and Nicotiana for directing the expression of S-locus genes.
The DNA sequences required in cis for the expression of the SLG gene lie within 3.65 kb upstream of the gene's coding region. Promoter activity is detected in both pollen and pistil of transformed Brassica and Nicotiana plants and follows the temporal, spatial, and developmental-regulated expression pattern noted above.